The present invention relates to DNA sequences which on the codogenic strand, code plant debranching enzymes whose transcripts formed in transgenic plants code new proteins with the enzymatic activity of debranching enzymes which in transgenic plants reduce the degree of branching of amylopectin starch. (The invention also Relates) to DNA sequences which on the codogenic strand code plant debranching enzymes whose transcripts formed in transgenic plants prevent the synthesis of proteins with the enzymatic activity of debranching enzymes, which in transgenic plants increases the degree of branching of amylopectin starch, and also to recombinant plasmids on which these DNA sequences are localized and which can be introduced into plant cells and plants.
The invention also relates to a process for the production of plants changed by genetic engineering whose amylopectin starch is modified, and to the modified starch obtainable from these plants.
Polysaccharides such as starch are, alongside along with oils, fats and proteins, essential renewable raw materials from plants.
A decisive factor which stands in the way of the use of renewable raw materials is the lack of substances which precisely meet the requirements of the chemical industry in regard to form, structure, or other physico-chemical parameters. In order to make the application of renewable raw materials feasible in as many fields of use as possible, it is particularly important to achieve a great material diversity. In regard to polysaccharides, this means that, for example, as many different forms of starch must be provided as possible. This necessitates considering both strongly branched forms which are characterized by a high surface reactivity in their chemical properties, and mildly branched types which are distinguished by a high uniformity of structure. Uniformity of structure is an important prerequisite for highly efficient reaction control during chemical syntheses.
Although starch is a polymer comprising chemically uniform basic components, the glucose molecules, it is a complex mixture of very different molecule forms which differ in respect to their degree of polymerization and the occurrence of branchings of the glucose chains. Starch is therefore not a uniform raw material. In particular, a distinction is drawn between amylose starch, an essentially unbranched polymer comprising .alpha.-1,4 glycosidically linked glucose molecules, and amylopectin starch, which for its part is a complex mixture of differently branched glucose chains. The branchings come about through the occurrence of additional .alpha.-1,6 glycosidic linkages.
In typical plants for starch production, such, for example maize or potato, the two forms of starch occur in a ratio of roughly 25 parts amylose to 75 parts amylopectin.
In regard to the uniformity of a basic substance, such as starch, for its application in the industrial sector, plants are needed which, for example, contain only the component amylopectin or plants which contain only the component amylose. In regard to the versatility of the raw material starch, plants are needed which show forms of amylopectin with differently marked branching. There is thus a great interest in enzymes of the starch metabolism which can modify the degree of branching of the starch molecules, or in gene sequences which can be used for the genetically changing plants so as to be able to synthesize different forms of starch in plants.
It is already known that for certain plant species, for example maize, plant types which contain only amylopectin can be produced by mutagenesis in which individual genes of the plant are inactivated. For potato, a genotype which forms no amylose was likewise produced by chemical mutagenesis with a haploid line (Hovenkamp-Hermelink et al., 1987, Theor Appl Genet 75: 217-221). Haploid lines, or the homozygotic diploid or tetraploid lines developed from them, are not usable in agriculture, however. The mutagenesis technique is not applicable to the agriculturally interesting heterozygotically tetraploid lines, as an inactivation of all copies of a gene is technically not possible because of the presence of four different genotype copies. It is known from Visser et al. (1991, Mol Gen Genet 225: 289) that plant types which form substantially pure amylopectin starch can be produced by antisense inhibition of the gene for the starch granule-bound starch synthetase in potato.
A branching enzyme of the potato is known from WO 92/14827. This enzyme is known as the Q-enzyme of Solanum tuberosum. It is also known that, with the help of DNA sequences which contain the information for the branching enzyme of the potato described in WO 92/14827, transgenic plants can be produced in which the amylose/amylopectin ratio of the starch is changed.
While the occurrence of several Q-enzymes is known for other species, e.g. maize (Singh & Preiss, 1985, Plant Physiol 79: 34-40), it is not known whether, besides the branching enzyme of the potato known from WO 92/14827, other enzymes are involved in the synthesis of branched starch in potato. Besides the Q-enzymes which introduce branchings into starch molecules, enzymes occur in plants which dissolve branchings. These proteins, also known as debranching enzymes, are divided into three groups according to substrate specifity:
The pullulanases, which besides pullulane also use amylopectin, occur in microorganisms, e.g. Klebsiella, and plants. In plants, they are also called R-enzymes. The isoamylases, which do not work with pullulane, but do with glycogen and amylopectin, likewise occur in microorganisms and plants. An isoamylase of maize is described by Manners & Rowe (1969, Carbohydr. Res. 9: 107), and Ishizaki et al. (1983, Agric Biol Chem 47: 771-779) describe an isoamylase of potato. The amylo-1,6-glucosidases are described in mammals and yeasts and use limiting dextrins as substrates.
Besides five endo- and two exoamylases, Li et al. (1992, Plant Physiol 98: 1277-1284) detected only one debranching enzyme of the pullulanase type in sugar beet. This enzyme, which has a size of ca. 100 kDa and a pH optimum of 5.5, is localized in the chloroplast.
Ludwig et al. (1984, Plant Physiol 74: 856-861) describe a debranching enzyme from spinach which uses pullulane as a substrate but which displays an activity three times lower upon reaction with amylopectin.
In the case of the agriculturally important starch-storing cultivated plant the potato, the activity of a debranching enzyme was investigated in 1951 by Hobson et al. (1951, J Chem Soc 1451). It was demonstrated that the corresponding enzyme, unlike the Q-enzyme, does not possess a chain-extending activity and merely hydrolyses .alpha.-1,6 glycosidic bonds. However, it was possible neither to characterize the enzyme more precisely nor to describe DNA sequences which code a protein with the enzymatic activity of a debranching enzyme.
To date, no DNA sequences are known which code a protein with the enzymatic activity of a debranching enzyme from plants which, upon introduction into the plant genome, change the metabolism of the plant in such a way that the degree of branching of the amylopectin starch is increased or reduced.